All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
The 65-kDa heat shock protein (HSP65) of the Mycobacterium tuberculosis (MT) plays a significant role in the pathogenesis of autoimmune arthritis. Its effect is well exemplified in the experimental model of adjuvant arthritis (AA). AA can be induced in susceptible, inbred strains of rats such as Lewis or Wistar, by intracutaneous inoculation of heat killed mycobacteria suspended in Freund's adjuvant. AA can be passively transferred by a T-cell clone reactive to residues 180-188 of the HSP65 [Holoshitz, J. et al. Science 219:56-58 (1983)].
Evidence has been reported that protection from disease may be due to cellular responses to HSP65 [Lider, O. et al. Proc. Natl. Acad. Sci. 84:4577-4580 (1987); Moudgil, K. et al. J. Exp. Med. 185:1307-1316 (1997)], suggesting that this protein contains different epitopes which participate in both pathogenesis and acquisition of resistance. The inventors have previously shown that resistance to AA can also be conferred by antibodies against HSP65 and can be passively transferred by intravenous infusion of immunoglobulins from arthritis-resistant strains to arthritis-susceptible rats [Ulmansky, R. and Naparstek, Y. Eur. J. Immunol. 25:952-957 (1995)]. Further analysis defined the epitope specificity of the anti-HSP protective antibodies to amino-acid residues 31-46, designated as peptide-6 (also denoted by SEQ ID NO. 15) [Ulmansky, R. and Naparstek, Y. J. Immunol. 168: 6463-6469 (2002)]. Vaccination of Lewis rats with this peptide resulted in the production of antibodies against the whole molecule as well as resistance to disease induction.
The inventors have previously shown that polyclonal antibodies directed against peptide-6 stimulate Interleukin-10 (IL-10) production by peripheral blood mononuclear cells (PBMCs) [Ulmansky (2002) ibid.]. The anti-inflammatory cytokine IL-10 plays an important role in innate immunity mostly due to its inhibitory effects, which allow suppression of inflammatory responses. Monoclonal anti-peptide-6 antibodies generated by the inventors were shown to retain this protective effect by binding to PBMCs and stimulating IL-10 secretion from the cells.
The inventors have further shown that antibodies directed against peptide-6 interact not only with peptide-6, but moreover, they apparently cross react directly with a surface ligand on macrophages, and this interaction is the key to comprehension of the mechanism of action of these antibodies. Following binding of the anti-peptide-6 antibodies to macrophages, there is activation of a signal transduction pathway that leads to an increase in production and secretion of cytokines, specifically IL-10. As an anti-inflammatory cytokine, IL-10 attenuates and inhibits inflammatory processes, thereby leading to amelioration and treatment of an inflammatory disorder. This tilts the balance between pro-inflammatory Th1 cytokines, such as tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory Th2 cytokines, such as IL-10. Modulation of the Th1/Th2 balance towards a Th2 anti-inflammatory response using the anti-peptide-6 antibodies is therefore applicable in the treatment of inflammatory disorders.
However, the administration of rodent antibodies or antibody fragments has had drawbacks that limit their applicability in humans due, in part, to immunogenicity of the antibodies or fragments thereof. To overcome the undesirable properties of rodent antibodies, humanized antibodies have been developed by replacing the framework regions, excluding the antigen-binding site, with regions of a human antibody. A method frequently used for preparing such humanized antibodies is based on selection of a gene encoding a human antibody showing the closest sequence similarity to the mouse antibody and replacing only the complementarity determining region (CDR) of the human antibody with that of the mouse antibody by way of a CDR grafting method. The humanized antibody has the advantage of reducing the in vivo immune response. However, when only the CDR is grafted on the human antibody, its selectivity and reactivity are often compromised.
Moreover, although the techniques of humanization have provided antibodies which are generally well tolerated when administered to man and generally have less immunogenicity than non-human monoclonal antibodies. Several antibodies generated by these techniques have been shown to elicit immunogenicity in patients even where the genetic origins of such antibodies are human. Such induction of immunogenicity is likely to result from the presence, within the antibody variable region, of tracts of non-self amino acid sequences which, in some cases, can create T cell epitopes which induce T cell responses resulting in immunogenicity.
The potential use of anti-peptide-6 antibodies as immuno-modulating agents requires production of a humanized antibody having reduced antigenic potential. There is therefore a need to produce highly specific humanized anti-peptide-6 antibodies having a high primarily sequences of human origin and avoiding the creation of sequences which might induce T cell responses.
It is therefore an object of the invention to provide such humanized anti-peptide-6 antibodies for modulating the Th1/Th2 balance in a subject suffering from an immune-related disorder.
These and other objects of the invention will become apparent as the description proceeds.